Cyloxygenase-2 (COX-2), the enzyme responsible for production of prostaglandins, has been identified as an important contributor to brain damage following hypoxia-ischemia. Unfortunately, COX-2 inhibitors have been reported to cause cerebral and cardiac thrombosis in patients, making COX-2 inhibition less attractive as a therapy for patients recovering from ischemic events. Thus, it is important to understand the mechanisms of COX-2 mediated neuronal injury so that more targeted therapies can be developed. Recently, downstream products of prostaglandins, specifically cyclopentenone prostaglandins have been identified as potential contributors to ischemic injury. The cyclopentenone prostaglandins are highly electrophilic and can form covalent bonds with exposed sulfhydryl groups of proteins. This chemical reaction can exacerbate recovery in reperfusing neurons by targeting nascent and denatured proteins thus contributing to endoplasmic reticulum (ER) stress. ER stress has been reported as an important component of ischemic injury. Further, cyclopentenones have been reported to bond and inactivate key component of the ER stress response. The ER stress response involves: 1) temporary cessation of translation of most mRNA (limiting the load of newly-formed, unfolded proteins) 2) selective translation of chaperone proteins to facilitate protein folding, and 3) recruitment of protein degradation pathways, including ubiquitination, the proteosome, and autophagy, to clear misfolded proteins. Cyclopentenone prostaglandins have been shown to bind with and inactivate key components of the ER stress response including thioredoxin, thioredoxin reductase, proteosome subunits, and ubiquitin hydrolase. We have identified a potential new target of this pathway, namely protein disulfide isomerase (PDI), an abundant ER protein responsible for proper folding of disulfide bonds in nascent or misfolded proteins. We are also the first to measure a cyclopentenone in post-ischemic brain tissue. We have been able to detect this highly reactive compound by using mass spectrometry in post-natal day 17 (PND 17) rat brains recovering from asphyxial cardiac arrest. The PND 17 rat is attractive as a model for this inquiry because it has very high constitutive expression of COX-2 and the insult mimics the most common cause of pediatric cardiac arrest (asphyxia) using a rat with developmental qualities similar to a toddler. Accordingly, we propose a series of experiments designed to investigate the hypothesis that COX-2 derived cyclopentenones worsen ER stress, in part by inhibiting PDI, in PND17 rats with asphyxial cardiac arrest. These experiments have the potential to identify new strategies for therapy in children suffering from brain injury following cardiac arrest. PUBLIC HEALTH RELEVANCE: This project will investigate a novel mechanism of brain injury following asphyxial cardiac arrest. We will use a rodent model mimicking pediatric cardiac arrest secondary to mechanisms such as drowning. The project has potential to identify therapies for brain injury in children suffering from cardiac arrest.